This invention relates to the field of plant molecular biology, more specifically to the invention of a DNA construct for conferring improved insect resistance to a corn plant, and even more specifically to an insect resistant corn plant transformation event MON810 and progeny thereof, and to assays for detecting the presence of MON810 DNA in a sample and compositions thereof.
As used herein, the term xe2x80x9ceventxe2x80x9d refers to a plant produced by transformation and regeneration of a single plant cell with heterologous DNA, i.e., a DNA construct that includes a transgene of interest. Normally, transformation of plant tissue produces multiple events, each of which represent insertion of a DNA construct into a different location in the genome each of a plant cell. Based on the expression of the transgene or other desirable characteristics, a particular event is selected.
The expression of foreign genes in plants is known to be influenced by their chromosomal position, perhaps due to chromatin structure (e.g., heterochromatin) or the proximity of transcriptional regulation elements (e.g., enhancers) close to the integration site (Weising et al., 1988, xe2x80x9cForeign Genes in Plants,xe2x80x9d Ann. Rev. Genet. 22:421-477). For this reason, it is often necessary to screen a large number of events in order to identify an event characterized by optimal expression of an introduced gene of interest. For example, it has been observed in plants and in other organisms that there may be wide variations in levels of expression of a heterologous gene introduced into the chromosome of a plants"" genome among individually selected events. There may also be differences in spatial or temporal patterns of expression, for example, differences in the relative expression of a transgene in various plant tissues, that may not correspond to the patterns expected from transcriptional regulatory elements present in the introduced gene construct. For this reason, it is common to produce hundreds to thousands of different events and screen those events for a single event that has desired transgene expression levels and patterns for commercial purposes. An event that has desired levels or patterns of transgene expression is useful for introgressing the transgene into other genetic backgrounds by sexual outcrossing using conventional breeding methods. Progeny of such crosses maintain the transgene expression characteristics of the original transformant. This strategy is used to ensure reliable gene expression in a number of varieties that are well adapted to local growing conditions.
It would be advantageous to be able to detect the presence of a particular event in order to determine whether progeny of a sexual cross contain a transgene of interest. In addition, a method for detecting a particular event would be helpful for complying with regulations requiring the pre-market approval and labeling of foods derived from recombinant crop plants, for example. It is possible to detect the presence of a transgene by any well known nucleic acid detection method including but not limited to thermal amplification (PCR(trademark)) or DNA hybridization using nucleic acid probes. Typically, for the sake of simplicity and uniformity of reagents and methodologies for use in detecting a particular DNA construct that has been used for transforming various plant varieties, these detection methods generally focus on frequently used genetic elements, such as promoters, terminators, marker genes, etc., because for many DNA constructs, the coding sequence region is interchangeable. As a result, such methods may not be useful for discriminating between constructs that differ only with reference to the coding sequence. In addition, such methods may not be useful for discriminating between different events, particularly those produced using the same DNA construct unless the sequence of chromosomal DNA adjacent to the inserted heterologous DNA (xe2x80x9cflanking DNAxe2x80x9d) is known. An event-specific thermal amplification (PCR(trademark)) assay is discussed, for example, by Windels et al. (Med. Fac. Landbouww, Univ. Gent 64/5b:459-462, 1999), who identified glyphosate tolerant soybean event 40-3-2 using a thermal amplification primer set spanning the junction between the insert and flanking DNA. Specifically, one primer was comprised of sequence from within the insert and a second primer was comprised of sequence from flanking DNA.
The inventor herein discloses novel and useful isolated nucleic acid sequences, as well as methods for detecting these nucleic acids in a biological sample, and kits comprising the reagents necessary for use in detecting these nucleic acids in a biological sample.
The inventor herein has discovered a corn event, MON810, that is resistant to Lepidopteran insect infestation, and also provides compositions and methods for detecting the presence of genomic DNA from this event in a biological sample.
According to one aspect of the invention, DNA sequences are provided that comprise at least one junction sequence of corn event MON810 selected from the group consisting of 5xe2x80x2-ACATCCTTTGCCATTGCCCA-3xe2x80x2 (SEQ ID NO:1) and 5xe2x80x2-GAACGAGGACTTTCGGTAGC-3xe2x80x2 (SEQ ID NO:2) and complements thereof (i.e., one or both junction sequences), wherein a junction sequence spans the junction between heterologous DNA inserted into the corn genome and DNA from the corn genome flanking the insertion site and is diagnostic for the event. Included are DNA sequences that comprise at least 10 or more (e.g., 15, 25, 50) nucleotides of insert sequence from corn event MON810 and similar length of flanking DNA from corn event MON810. Also included are DNA sequences which comprise 15 or more nucleotides of contiguous insert sequence from corn event MON810 and at least one nucleotide of flanking DNA from corn event MON810 adjacent to the insert sequence. Such DNA sequences are diagnostic for corn event MON810. Nucleic acid amplification of genomic DNA from the event produces an amplicon comprising such diagnostic DNA sequences.
According to another aspect of the invention, corn plants comprising such DNA sequences and seed from such corn plants are provided.
According to another aspect of the invention, flanking sequence primers for detecting corn event MON810 are provided. Such flanking sequence primers comprise an isolated nucleic acid sequence comprising at least 15 contiguous nucleotides from nucleotides 1-244 of SEQ ID NO:3 (arbitrarily designated herein as the 5xe2x80x2 flanking sequence), at least 15 contiguous nucleotides from nucleotides 274-879 of SEQ ID NO:4 (arbitrarily designated herein as the 3xe2x80x2 flanking sequence), or the complements thereof.
According to another aspect of the invention, primer sets that are useful for nucleic acid amplification, for example, are provided. Such primer sets comprise a primer comprising a nucleotide sequence of at least 10-15 contiguous nucleotides in length which is or is complementary to one of the above-described genomic flanking sequences (SEQ ID NO:3 or SEQ ID NO:4) and a second primer comprising at least an isolated 10-15 contiguous nucleotides of heterologous DNA inserted into the plant DNA sequence, i.e., the DNA sequence inserted into the recombinant corn plant event MON810. The second primer is preferably a polynucleotide sequence which is or is complementary to the insert sequence adjacent to the plant genomic flanking DNA sequence as set forth in SEQ ID NO:3 from nucleotide position 245 through 566 and in SEQ ID NO:4 from nucleotide position 1 through 273. Of course, it is well within the skill in the art to obtain additional sequence further out into the genome sequence flanking either end of the inserted heterologous DNA sequence for use as a primer sequence that can be used in such primer pairs for amplifying the sequences that are diagnostic for the MON810 corn event. For the purposes of this disclosure, the phrase xe2x80x9cfurther out into the genome sequence flanking either end of the inserted heterologous DNA sequencexe2x80x9d refers specifically to a sequential movement away from the ends of the inserted heterologous DNA sequence, the points at which the inserted DNA sequence is adjacent to native genomic DNA sequence, and out into the genomic DNA of the particular chromosome into which the heterologous DNA sequence was inserted. Preferably, a primer sequence corresponding to or complementary to a part of the insert sequence should prime the transcriptional extension of a nascent strand of DNA or RNA toward the nearest flanking sequence junction. Consequently, a primer sequence corresponding to or complementary to a part of the genomic flanking sequence should prime the transcriptional extension of a nascent strand of DNA or RNA toward the nearest flanking sequence junction. A primer sequence can be, or can be complementary to, a heterologous DNA sequence inserted into the chromosome of the plant, or a genomic flanking sequence. One skilled in the art would readily recognize the benefit of whether a primer sequence would need to be, or would need to be complementary to, the sequence as set forth within the inserted heterologous DNA sequence or as set forth in SEQ ID NO:3 or SEQ ID NO:4 depending upon the nature of the product desired to be obtained through the use of the nested set of primers intended for use in amplifying a particular flanking sequence containing the junction between the genomic DNA sequence and the inserted heterologous DNA sequence.
According to another aspect of the invention, methods of detecting the presence of DNA corresponding to the corn event MON810 in a biological sample are provided. Such methods comprise: (a) contacting the sample comprising DNA with a primer set that, when used in a nucleic-acid amplification reaction with genomic DNA from corn event MON810, produces an amplicon that is diagnostic for corn event MON810; (b) performing a nucleic acid amplification reaction, thereby producing the amplicon; and (c) detecting the amplicon.
According to another aspect of the invention, one method of detecting the presence of a DNA corresponding to the MON810 event in a biological sample comprises: (a) contacting the biological sample comprising DNA or RNA with a probe that hybridizes under stringent hybridization conditions with genomic DNA from corn event MON810 and does not hybridize under the same stringent hybridization conditions with DNA from a control corn plant; (b) subjecting the sample and probe to stringent hybridization and wash conditions; and (c) detecting hybridization of the probe to the DNA. Detection can be by any means well known in the art including but not limited to fluorescent, chemiluminescent, radiological, immunological, or otherwise. In the case in which hybridization is intended to be used as a means for amplification of a particular sequence to produce an amplicon which is diagnostic for the MON810 maize event, the production and detection by any means well known in the art of the amplicon is intended to be indicative of the intended hybridization to the target sequence where one probe or primer is utilized, or sequences where two or more probes or primers are utilized. The term xe2x80x9cbiological samplexe2x80x9d is intended to comprise a sample that contains or is suspected of containing a nucleic acid comprising from between five and ten nucleotides either side of the point at which one or the other of the two terminal ends of the inserted heterologous DNA sequence contacts the genomic DNA sequence within the chromosome into which the heterologous DNA sequence was inserted, herein also known as the junction sequences. In addition, the junction sequence comprises as little as two nucleotides: those being the first nucleotide within the flanking genomic DNA adjacent to and covalently linked to the first nucleotide within the inserted heterologous DNA sequence.
Another aspect of the present invention is methods and compositions for detecting the presence of a target site, i.e., at least one of the junctions of the insert DNA with the genomic DNA of the corn plant in MON810, in a sample of nucleic acid derived from or obtained from the genome of the MON810 event, using a variety of detection methods including TAQMAN (Perkin Elmer), thermal amplification, ligase chain reaction, southern hybridization, ELISA methods, and colorimetric and fluorescent detection methods. In particular the present invention provides for kits for detecting the presence of the target sequence, i.e., at least one of the junctions of the insert DNA with the genomic DNA of the corn plant in MON810, in a sample containing genomic nucleic acid from MON810. The kit is comprised of at least one polynucleotide capable of binding to the target site or substantially adjacent to the target site and at least one means for detecting the binding of the polynucleotide to the target site. The detecting means can be either fluorescent, chemiluminescent, colorimetric, or isotopic and can be coupled at least with immunological methods for detecting the binding. A kit is also envisioned which can detect the presence of the target site in a sample, i.e., at least one of the junctions of the insert DNA with the genomic DNA of the corn plant in MON810, taking advantage of two or more polynucleotide sequences which together are capable of binding to nucleotide sequences adjacent to or within about 100 base pairs, or within about 200 base pairs, or within about 500 base pairs or within about 1000 base pairs of the target sequence and which can be extended toward each other to form an amplicon which contains at least the target site.
According to another aspect of the invention, methods of detecting the presence of DNA corresponding to the maize event MON810 in a sample are provided. Such methods comprise: (a) contacting the sample comprising DNA with a primer set that, when used in a nucleic-acid amplification reaction with genomic DNA from maize event MON810, produces an amplicon that is diagnostic for maize event MON810; (b) performing a nucleic acid amplification reaction, thereby producing the amplicon; and (c) detecting the amplicon.
According to another aspect of the invention, a kit is provided for the detection of maize event MON810. The kit includes at least one DNA sequence of sufficient length of polynucleotides which is or is complementary to SEQ ID NO:1 or SEQ ID NO:2, wherein the DNA sequences are useful as primers or probes that hybridize to isolated DNA from maize event MON810 or its progeny, and which, upon hybridization to a nucleic acid sequence in a sample followed by detection of the hybridization to the target sequence, are diagnostic for the presence of nucleic acid sequences from maize event MON810 in said sample.
The foregoing and other aspects of the invention will become more apparent from the following detailed description and accompanying drawings.
The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. Definitions of common terms in molecular biology may also be found in Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994. The nomenclature for DNA bases as set forth at 37 CFR xc2xa71.822 is used.
As used herein, the term xe2x80x9ccomprisingxe2x80x9d means xe2x80x9cincluding but not limited toxe2x80x9d.
Event
As used herein a transgenic xe2x80x9ceventxe2x80x9d refers to a recombinant plant produced by transformation of plant cells with heterologous DNA, i.e., a nucleic acid construct that includes a transgene of interest, regeneration of a population of plants resulting from the insertion of the transgene into the genome of the plant, and selection of a particular plant characterized by insertion into a particular genome location. The term xe2x80x9ceventxe2x80x9d refers to the original transformant and progeny of the transformant that include the heterologous DNA. The term xe2x80x9ceventxe2x80x9d also refers to progeny produced by a sexual outcross between the transformant and another variety that include the heterologous DNA. Even after repeated backcrossing to a recurrent parent, the inserted DNA and flanking DNA from the transformed parent is present in the progeny of the cross at the same chromosomal location. The term xe2x80x9ceventxe2x80x9d also refers to DNA from the original transformant comprising the inserted DNA and flanking sequence immediately adjacent to the inserted DNA that would be expected to be transferred to a progeny that receives inserted DNA including the transgene of interest as the result of a sexual cross of one parental line that includes the inserted DNA (e.g., the original transformant and progeny resulting from selfing) and a parental line that does not contain the inserted DNA.
Probes and Primers
A xe2x80x9cprobexe2x80x9d is an isolated nucleic acid to which is attached a conventional detectable label or reporter molecule, e.g., a radioactive isotope, ligand, chemiluminescent agent, or enzyme. Such a probe is complementary to a strand of a target nucleic acid, in the case of the present invention, to a strand of genomic DNA from corn event, MON810 (whether from a corn plant or from a sample that includes DNA from the event). Probes according to the present invention include not only deoxyribonucleic or ribonucleic acids but also polyamides and other probe materials that bind specifically to a target DNA sequence and can be used to detect the presence of that target DNA sequence.
xe2x80x9cPrimersxe2x80x9d are isolated nucleic acids that are annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, then extended along the target DNA strand by a polymerase, e.g., a DNA polymerase. Primer pairs or sets can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR(trademark)), also known as thermal amplification methods, or other conventional nucleic-acid amplification methods.
Probes and primers are generally between 10 and 15 nucleotides or more in length, preferably 20 nucleotides or more, more preferably 25 nucleotides, and most preferably 30 nucleotides or more. Such probes and primers hybridize specifically to a target sequence under high stringency hybridization conditions. Preferably, probes and primers according to the present invention have complete sequence complementarity with the target sequence, although probes differing from the target sequence and which retain the ability to hybridize to target sequences may be designed by conventional methods.
Methods for preparing and using probes and primers are described, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989 (hereinafter, xe2x80x9cSambrook et al., 1989xe2x80x9d); Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates) (hereinafter, xe2x80x9cAusubel et al., 1992); and Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990. PCR(trademark)-primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, (copyright) 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.).
Primers and probes based on the flanking DNA and insert sequences disclosed herein can be used to confirm (and, if necessary, to correct) the disclosed sequences by conventional methods, e.g., by re-cloning and sequencing such sequences.
Nucleic-Acid Hybridization; xe2x80x9cStringent Conditionsxe2x80x9d; xe2x80x9cSpecificxe2x80x9d
The nucleic-acid probes and primers of the present invention hybridize under stringent conditions to a target DNA sequence. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA from a transgenic event in a sample.
The term xe2x80x9cstringent conditionsxe2x80x9d is functionally defined with regard to the hybridization of a nucleic-acid probe to a target nucleic acid (i.e., to a particular nucleic-acid sequence of interest) by the specific hybridization procedure discussed in Sambrook et al., 1989, at 9.52-9.55. See also, Sambrook et al., 1989 at 9.47-9.52, 9.56-9.58 herein incorporated by reference in its entirety; Kanehisa, (Nucl. Acids Res. 12:203-213, 1984, herein incorporated by reference in its entirety); and Wetmur and Davidson, (J. Mol. Biol. 31:349-370, 1988, herein incorporated by reference in its entirety). Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of DNA fragments. Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g., one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50xc2x0 C. to about 70xc2x0 C. One stringent condition, for example, is to wash the hybridization filter at least twice with high-stringency wash buffer (0.2xc3x97SSC, 0.1% SDS, 65xc2x0 C.). Appropriate stringency conditions which promote DNA hybridization, for example, 6.0xc3x97sodium chloride/sodium citrate (SSC) at about 45xc2x0 C., followed by a wash of 2.0xc3x97SSC at 50xc2x0 C., are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0xc3x97SSC at 50xc2x0 C. to a high stringency of about 0.2xc3x97SSC at 50xc2x0 C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22xc2x0 C., to high stringency conditions at about 65xc2x0 C. Both temperature and salt may be varied, or either the temperature or the salt concentration may be held constant while the other variable is changed. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand. Detection of DNA sequences via hybridization is well-known to those of skill in the art, and the teachings of U.S. Pat. Ser. Nos. 4,965,188 and 5,176,995 are exemplary of the methods of hybridization analyses.
Regarding the amplification of a target nucleic- acid sequence (e.g., by thermal amplification or PCR) using a particular amplification primer pair, xe2x80x9cstringent conditionsxe2x80x9d are conditions that permit the primer pair to hybridize only to the target nucleic-acid sequence to which a primer having the corresponding wild-type sequence (or its complement) would bind and preferably to produce a unique amplification product, the amplicon.
The term xe2x80x9cspecific for (a target sequence)xe2x80x9d indicates that a probe or primer hybridizes under stringent hybridization conditions only to the target sequence in a sample comprising the target sequence.
As used herein, xe2x80x9camplified DNAxe2x80x9d or xe2x80x9campliconxe2x80x9d refers to the product of nucleic acid amplification of a target nucleic acid sequence that is part of a nucleic acid template. For example, to determine whether the corn plant resulting from a sexual cross contains a transgenic event, genomic DNA from a corn plant may be subjected to nucleic acid amplification using a primer pair that includes a primer derived from flanking sequence in the genome of the plant adjacent to the insertion site of inserted heterologous DNA and a second primer derived from the inserted heterologous DNA to produce an amplicon that is diagnostic for the presence of the event. The amplicon is of a length and has a sequence that is diagnostic for the event. Alternatively, a primer pair can be derived from flanking sequence on both sides of the inserted DNA so as to produce an amplicon that includes the entire insert.
Nucleic acid amplification can be accomplished by any of the various nucleic acid amplification methods known in the art, including the polymerase chain reaction (PCR). A variety of amplification methods are known in the art and are described, inter alia, in U.S. Pat. Nos. 4,683,195 and 4,683,202 and in PCR Protocols: A Guide to Methods and Applications, ed. Innis et al., Academic Press, San Diego, 1990. Any well known method for nucleic acid amplification may be used in the practice of the present invention. The sequence of the heterologous DNA insert or flanking sequence from corn MON810 event can be verified (and corrected if necessary) by amplifying such sequences from the event using primers derived from the sequences provided herein followed by standard methods of DNA sequencing of the PCR amplicon or of the cloned DNA molecule.
The amplicon produced by these methods may be detected by a plurality of techniques. Agarose gel electrophoresis and staining with ethidium bromide is a common well known method of detecting DNA amplicons. Another method is Genetic Bit Analysis (Nikiforov, et al. Nucleic Acid Res. 22:4167-4175, 1994) where an DNA oligonucleotide is designed which overlaps both the adjacent flanking genomic DNA sequence and the inserted DNA sequence. The oligonucleotide is immobilized in wells of a microtiter plate. Following thermal amplification of the region of interest (using one primer in the inserted sequence and one in the adjacent flanking genomic sequence), a single-stranded thermal amplification product can be hybridized to the immobilized oligonucleotide and serve as a template for a single base extension reaction using a DNA polymerase and labelled ddNTPs (dideoxynucleotides triphosphates) specific for the expected next base. Readout may be fluorescent or ELISA-based. A signal indicates presence of the insert/flanking sequence due to successful amplification, hybridization, and single base extension.
An additional method is the Pyrosequencing technique as described by Winge (Innov. Pharma. Tech. 00:18-24, 2000). In this method an oligonucleotide is designed that overlaps the adjacent genomic DNA and insert DNA junction. The oligonucleotide is hybridized to single-stranded thermal amplification product from the region of interest (one primer in the inserted sequence and one in the flanking genomic sequence) and incubated in the presence of a DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5xe2x80x2 phosphosulfate and luciferin. dNTPs (deoxynucleotide triphosphates) are added individually and the incorporation results in a light signal which is measured. A light signal indicates the presence of the transgene/flanking sequence due to successful amplification, hybridization, and single or multi-base extension.
Fluorescence Polarization as described by Chen, et al., (Genome Res. 9:492-498, 1999) is a method that can be used to detect the amplicon of the present invention. Using this method an oligonucleotide is designed which overlaps the genomic flanking and inserted DNA junction. The oligonucleotide is hybridized to a single-stranded thermal amplification product from the region of interest (one primer in the inserted DNA and one in the flanking genomic DNA sequence) and incubated in the presence of a DNA polymerase and a fluorescent-labeled ddNTP. Single base extension results in incorporation of the ddNTP. Incorporation can be measured as a change in polarization using a fluorometer. A change in polarization indicates the presence of the transgene/flanking sequence due to successful amplification, hybridization, and single base extension.
Taqman(copyright) (PE Applied Biosystems, Foster City, Calif.) is described as a method of detecting and quantifying the presence of a DNA sequence and is fully understood in the instructions provided by the manufacturer. Briefly, a FRET oligonucleotide probe is designed which overlaps the genomic flanking and insert DNA junction. The FRET probe and thermal amplification primers (one primer in the insert DNA sequence and one in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in cleavage and release of the fluorescent moiety away from the quenching moiety on the FRET probe. A fluorescent signal indicates the presence of the flanking/transgene sequence due to successful amplification and hybridization.
Molecular Beacons have been described for use in sequence detection as in Tyangi, et al. (Nature Biotech.14:303-308, 1996) Briefly, a FRET oligonucleotide probe is designed that overlaps the flanking genomic and insert DNA junction. The unique structure of the FRET probe results in it containing secondary structure that keeps the fluorescent and quenching moieties in close proximity. The FRET probe and thermal amplification primers (one primer in the insert DNA sequence and one in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Following successful thermal amplification, hybridization of the FRET probe to the target sequence results in the removal of the probe secondary structure and spatial separation of the fluorescent and quenching moieties. A fluorescent signal results. A fluorescent signal indicates the presence of the flanking/transgene sequence due to successful amplification and hybridization.
Detection Kit
As used herein, xe2x80x9cdetection kitxe2x80x9d refers to a kit used to detect the presence or absence of DNA from a MON810 event in a sample comprising nucleic-acid probes and primers of the present invention, which hybridize under stringent conditions to a target DNA sequence, and other materials necessary to enable nucleic acid hybridization or amplification methods. Alternatively, a detection kit may comprise materials necessary to enable one skilled in the art to perform methods similar to those described in PCT International Application WO 97/22719, incorporated herein by reference, to detect the presence or absence of DNA from a MON810 event in a sample.
The following examples are included to demonstrate examples of certain preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the invention, and thus can be considered to constitute examples of preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.